1. Field of the Invention
The present invention relates to a scanning laser microscope used to observe a biological sample and the like.
2. Description of the Prior Art
A scanning laser microscope is known as a microscope which optically slices a sample of a living cell or tissue without damaging the sample to obtain a plurality of two-dimensional tomograms and which obtains a three-dimensional image from a plurality of two-dimensional tomograms.
The scanning laser microscope is popularly used by making the above characteristic features efficient. In the fields of physiology, pharmacology, cell biology, and the like, a chemical or physical reaction and the manner of a change in a cell with respect to a stimulus or heat to the cell, e.g., a stimulus caused by an electric signal or a chemical stimulus caused by a drug or medicine are observed and recorded in detail. In the fields of morphology and embryology, the structure and shape of a cell or the manners of modification and movement with time are observed and recorded in detail.
The scanning laser microscope which observes a biological sample, a fluorescent reagent or a fluorescent protein which is introduced into the sample is excited by a laser beam, and an intensity of fluorescence generated by the excitation is measured to create an image. In this case, it is known that an intensity of fluorescence generated from a fluorescent material decreases with irradiation of the excitation light, i.e., a photo bleaching occurs. For this reason, a method which cuts unnecessary excitation light to prevent the sample from being bleached (faded) as much as possible is considered.
For example, variations per unit time of the two-dimensional or three-dimensional image is observed, a method which prevents a laser beam from unnecessarily being irradiated on a sample within an interval time from acquisition of an image to acquisition of the next image is employed. As this method, a shutter on an optical path is mechanically opened or closed, a laser beam is attenuated by using an optical filter or an acousto-optic filter, or output of a laser beam source is electrically switched on or off.
On the basis of these thoughts, in a microscope disclosed in Jpn. Pat. Appln. KOKAI Publication No. 9-189864, a region to be observed is set in an image of one frame, and a laser beam actually scans the region. In this case, irradiation of the laser beam is controlled in units of scanning pixels such that a transmission state xe2x80x9c1xe2x80x9d and a transmission state xe2x80x9c0xe2x80x9d are set in the set region and outside the set region, respectively. In this manner, a sample outside the region to be observed is prevented from being bleached. In an inspection method disclosed in Jpn. Pat. Appln. KOKAI Publication No. 2000-35400, while a laser scans a two-dimensional plane, intensity adjustment of the laser beam and selection of a wavelength are executed at respective positions by a preset procedure. In this manner, a laser beam having a desired wavelength and a desired intensity can be irradiated on a desired portion in the two-dimensional plane.
In recent years, a study in which a fluorescent bleaching phenomenon is positively used is developed. In this study, a specific region of a sample is bleached by irradiation of a laser beam. Thereafter, the movement, the diffusion coefficient, and the like of a fluorescent material in a restoring process of fluorescence in the bleached region are measured while a an observed region or irradiation conditions of the laser beam are changing. More specifically, immediately after a specific portion of a sample is bleached in an arbitrary shape, a measurement is performed such that a laser beam having a desired wavelength and a desired intensity is irradiated on the specific portion, a region around the specific portion, or both the regions.
However, in the Jpn. Pat. Appln. KOKAI Publication No. 9-189864 and the Jpn. Pat. Appln. KOKAI Publication No. 2000-35400, irradiation of the laser beam is controlled in a preset region and respective positions on the basis of determined conditions, and static observation is set as a main object. For this reason, as described above, the conventional methods cannot cope with a dynamic measurement with the passage of time which, after bleaching a specific region of a sample by irradiation of a laser beam, measures a restoring process of fluorescence of the bleached region while an observed region or irradiation conditions of the laser beam are changed.
It is an object of the present invention to provide a scanning laser microscope which can cope with a dynamic measurement with the passage of time.
According to the present invention, there is provided a scanning laser microscope including a laser generation unit which generates a laser beam, a light modulation unit which modulates the laser beam, a scanning unit which causes the laser beam to scan a sample, a light receiving unit which receives light from the sample, a control unit which controls the light modulation unit for each pixel of a scanning image acquired by light receiving of the light receiving unit, and a storage unit which stores a plurality of controlled patterns, the number of pixels of the patterns corresponding to the number of pixels of the scanning image, and in which modulation information of the laser beam is set for each pixel, wherein the control unit reads the control patterns stored in the storage unit, controls the light modulation unit on the basis of the modulation information of each pixel, and performs such control that the control patterns are changed at an arbitrary timing.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.